The techniques of rapid reaction kinetics and magnetic susceptibility will be used to study the thermodynamic and kinetic linkages between changes in protein conformation and iron electronic configuration in hemoglobin A and S, other mutant hemoglobins and various metalloproteins. To accomplish this a newly developed, unique, high resolution time-resolved magnetic susceptometer will be constructed in the first year of the grant. This instrument, which is based on recent advances in superconducting technology, will be one hundred times more sensitive than conventional susceptometers, provide micro-second time resolution and will make possible the study of the rapid kinetics of spin state changes in hemoglobins and other metalloproteins. This new Time-Resolved Magnetic Susceptibility (TREMS) method also can be used to investigate the influence of subunit association on spin state dynamics in dimeric and tetrameric hemeproteins as well as in extended assemblies such as hemoglobin S. While the mechanism of cooperative oxygen binding to hemoglobin is still not known, current proposals invoke coupling of spin state changes to conformational changes. We propose some critical tests of the Perutz localized "trigger" model versus the Hopfield distributed strain model for the mechanism of hemoglobin cooperativity. These magnetic studies will be carried out in parallel with absorption and resonance Raman spectroscopic studies of the same allosterically modifiable hemoglobins. Temperature-jump pulse relaxation methods will continue to be used to measure the elementary step rate constants for oxygen binding. A number of other metalloproteins will be studied to answer specific questions of electronic configuration and structure. For example, measuring the antiferromagnetic coupling in the invertebrate oxygen carriers hemerythrin and hemocyanin will elucidate the mode by which a single O2 is bound to two iron or copper atoms, respectively. The magnetic propoerties of cytochromes, ferredoxins, and hydrogenase will also be explored.